The goal of this work is to learn how the genes for several small nuclear RNAs are organized in the genome and how the structure and organization of these genes influence their expression. At least four different snRNA gene families will be studied: two from mouse and two from Drosophila cells. In each organism we will study the genes for U1 snRNA and a gene for another snRNA whose accumulation is modulated during heat shock or development. We plan to isolate clones of genomic DNA containing genes and pseudogenes for these RNAs. Appropriate regions of the clones will be sequenced. The number and distribution of DNA sequences in the genome complementary to coding regions and flanking regions will be determined by hybridization to size fractionated restriction fragments of cell DNA. Hybrids will be analyzed for complete homology with the cloned probes, using nuclease S1. The cloned snRNA genes will be used to study transcription of these RNAs in cell-free extracts, after injection into frog oocytes or after transformation of mouse cells. The extent of flanking sequences required for transcription will be determined by in vitro alteration of these DNA sequences. The role of other factors (e.g., proteins associated with snRNAs) for accurate and effective synthesis of these snRNAs will also be tested in cell-free systems. The functional expression of snRNAs will be studied in vivo by transfecting cells with snRNA genes whose coding sequences have been altered at specific sites in vitro. The results obtained should help us to understand the basic mechanisms involved in expression and control of eukaryotic genes and multi-gene families. They should also increase our understanding of the roles of snRNAs in control of gene expression and in cell growth, aging and metabolism.